The Expression of X-Linked Phosphoglycerate Kinase in the Early Mouse Embryo

Abstract. During early mouse embryogenesis, the activity of X-chromosomally linked maternal and paternal phosphoglycerate kinase (PGK-1) alleles was determined using electrophoretic separation of their gene products and a sensitive fluorometric enzyme assay. In the embryos collected from females homozygous for PGK-1^b mated with PGK-1^a males and vice versa, the paternally derived allozyme was first detected after implantation on day 6. Expression of the maternally inherited allele was studied in embryos from females heterozygous for PGK-1^b and PGK-1^a. From day 1 to day 4, the embryos maintained a constant ratio of enzyme activity of PGK-1B to PGK-1A. Prior to implantation of the embryos between day 4 and day 5, the activity ratio of the two PGK-1 allelic variants changed significantly due to the first appearance of newly synthesized PGK derived from the maternally inherited allele.
Our data demonstrate a temporal difference in the onset of PGK synthesis depending on whether this particular gene product is of maternal or paternal origin. Therefore, we conclude that the maternal PGK-1 locus is already activated during late preimplantation development whereas the paternally inherited gene locus remains silent at the preimplantation stage but is subsequently expressed at approximately the time of X-chromosomal inactivation.     

X-linked gene expression in metatherian fibroblasts: Evidence from theGpd andPgk-A loci of the Virginia opossum and the red-necked wallaby

Fibroblasts cultured from ear pinna biopsies of Virginia opossums (Didelphis virginiana) and red-necked wallabies (Macropus rufogriseus) were examined electrophoretically to determine the relative expression levels of the maternally and paternally derived alleles at X-linked, enzyme-coding loci. Only the maternally derived allele was expressed at thePgk-A locus in fibroblasts of heterozygousD. virginiana (M. rufogriseus not examined), but fibroblasts of both species exhibited evidence of paternal allele expression a t theGpd locus. Furthermore, the heterozygous G6PD phenotypes in both species were skewed in favor of the maternal gene product, as expected if the paternal allele is only partially (incompletely) expressed. ForM. rufogriseus this result is contrary to a previous finding which suggested equal expression of bothGpd alleles in cultured fibroblasts of this species. The present results suggest that X-linked genes in metatherian fibroblasts are subject to the same kind of determinate, paternal allele inactivation, incomplete at some loci, described previously for X-linked genes in adult tissues and that the pattern of paternal X-linked gene expression in these cells is independent of the patterns in the tissues from which the fibroblasts are derived.The work was supported in part by grants from the National Institutes of Health (Biomedical Research Support Grant RR-05519) and the National Science Foundation (DCB 8516949).     

Synchronous activation of both parental alleles at the 6-PGD locus of Japanese quail embryos

The gene locus for the enzyme 6-phosphogluconate dehydrogenase belongs to that part of the genome which is activated at the beginning of embryonic development. The present experiment, utilizing three alleles at this autosomally inherited locus of the Japanese quail, was designed to show whether exhaustion of maternally stored 6-PGD is followed by maternally hemizygous de novo synthesis of the same enzyme. 6-PGD phenotypes of early embryos resulting from the mating between a male homozygous for one allele and a female heterozygous for two other alleles were examined by starch gel electrophoresis. The result showed that the maternally stored 6-PGD is exhausted before the twenty-fourth hour of incubation. This is followed by synchronous activation of both parental alleles. Previous studies on the development of various interspecific crosses have revealed that, at all loci studied, the activation of the maternally derived allele preceded that of the paternally derived allele. The present experiment reveals that preferential activation of maternally derived alleles need not be a rule of development.This work was supported in part by a grant (CA-05138) from the National Cancer Institute, U.S. Public Health Service, and in part by a research fund established in honor of General James H. Doolittle. Contribution No. 6-68, Department of Biology, City of Hope Medical Center.     

Asynchronous activation of parental alleles at the tissue-specific gene loci observed on hybrid trout during early development

By using three genetic markers, the asynchronous activation of parental alleles during embryonic development was studied on interspecific hybrids between the male brown trout and the female rainbow trout. The genetic markers used were lactate dehydrogenase (LDH) A² as well as C subunits and alcohol dehydrogenase (ADH) subunits. Starch gel electrophoresis was utilized. The presence of LDH A² subunits was detected from a very early stage of development in whole embryo extracts. However, up to a few days after hatching (63rd day), A² subunits present were exclusively of the A^2R type derived from the rainbow mother. The coexistence of the products of both parental alleles at this gene locus became evident only at the 70th day. The LDH C subunits which occur only in the retina of the eye as well as the ADH subunits which occur only in liver made their first appearances at the 95th and 150th days, respectively. At these dates, only the maternally derived rainbow subunits were detectable. It took another 40 days for the paternally derived brown subunits to make their appearance.In Duarte, this work was supported in part by PHS grant CA-05138 from the National Cancer Institute, U.S. Public Health Service.     

Allelic inhibition at the autosomally inherited gene locus for liver alcohol dehydrogenase in chicken-quail hybrids

At the gene locus for liver alcohol dehydrogenase (ADH) of the Japanese quail, three alleles which give electrophoretic variants, A, B, and C, exist. This enzyme is autosomally inherited. Allelic polymorphism was not observed in the chicken, but the wild-type ADH of the chicken can readily be distinguished from A, B, and C of the quail by starch gel electrophoresis. In the development of both species, ADH activity reached a near adult level at about the nineteenth day (a few days after hatching in the quail and a few days before hatching in the chicken). Chicken-quail hybrids at the day of hatching (nineteenth day) revealed the presence of maternally derived quail ADH only, and their ADH activities were about half that of both parental species. Those hybrids which received either A or C allele from the mother quail showed three bands of ADH at the third day after hatching. The chicken and quail alleles began to function in synchronous harmony. One 3-day-old and two adult hybrids which received B allele from the quail, however, still revealed complete absence of the paternally derived chicken ADH.This work was supported in part by a grant (CA-05138) from the National Cancer Institute, U.S. Public Health Service, and in part by a research fund established in honor of General James H. Doolittle. Contribution No. 20-67, Department of Biology, City of Hope Medical Center.Dr. Eduardo Castro-Sierra is a fellow of the Institute for Advanced Learning of the City of Hope Medical Center.     

Parental modifiers,antisense transcripts and loss of imprinting

The kinship theory of genomic imprinting has explained parent-specific gene expression as the outcome of an evolutionary conflict between the two alleles at a diploid locus of an offspring over how much to demand from parents. Previous models have predicted that maternally derived (madumnal) alleles will be silent at demand-enhancing loci, while paternally derived (padumnal) alleles will be silent at demand-suppressing loci, but these models have not considered the evolution of trans-acting modifiers that are expressed in parents and influence imprinted expression in offspring. We show that such modifiers will sometimes be selected to reactivate the silent padumnal allele at a demand-suppressing locus but will not be selected to reactivate the silent madumnal allele at a demand-enhancing locus. Therefore, imprinting of demand-suppressing loci is predicted to be less evolutionarily stable than imprinting of demand-enhancing loci.     

Reversion at the HIS1 Locus of Yeast

The his1 gene (chromosome V) of Saccharomyces cerevisiae specifies phosphoribosyl transferase (E.C.2.4.2.17), the first enzyme of histidine biosynthesis. This hexameric enzyme has both catalytic and regulatory functions. The spontaneous reversion rates of seven his1 mutations were studied. The reversion rates of the alleles at the proximal end of the locus (relative to the centromere) were about 50-fold higher than distal alleles. Spontaneous reversion to prototrophy was studied in diploids homoallelic for each of the seven his1 mutations. Based on tetrad analysis, the prototrophy revertants could be assigned to three classes: (1) revertant tetrads that carried a prototrophic allele indistinguishable from wild type; (2) revertant tetrads that carried a prototrophic allele characterized by histidine excretion and feedback resistance; and (3) revertant tetrads that did not contain a prototrophic spore, but rather a newly derived allele that complemented the original allele intragenically. Four of the seven his1 mutations produced the excretor revertant class, and two mutations produced the complementer revertant class. The significance of these findings to our understanding of gene organization and the catalytic and regulatory functions of gene products are discussed.     

Expression of X-linked phosphoglycerate kinase in early mouse embryos homozygous at the Xce locus

The expression of maternally derived X-chromosomal Pgk-1 alleles was investigated in oocytes and early embryos of mice carrying different alleles (Xcea, Xcec) of the X-chromosome controlling element (Xce) locus. Pgk-1 allelic expression was determined by measuring their gene products using Cellogel electrophoresis and a sensitive fluorimetric enzyme assay. In addition to the already existing mouse strain of the genotypes Pgk-1a Xcec and Pgk-1b Xcea, a new line was bred carrying the combination Pgk-1b Xcec. The X chromosomes carrying the combinations Pgk-1a Xcec and Pgk-1b Xcec were of feral origin, whereas Pgk-1b Xcea was derived from a laboratory line. Our results using Xcec homozygous females confirm that maternal Pgk-1 is already expressed on day 4 of embryogenesis, thus substantiating data previously obtained using Xcea/Xcec heterozygous females. This finding also demonstrates that the timing of reactivation of maternal Pgk-1 is not influenced by the Xce locus. Furthermore, we found that oocytes from Xcec homozygous females have a balanced PGK-1 A/PGK-1 B allozyme ratio (50:50), whereas in oocytes obtained from Xcea/Xcec heterozygotes, the PGK-1 allozyme ratio is about 60:40. In tissues of adult Xce homozygous females, the PGK-1 allozymes are also balanced, whereas in Xcea/Xcec heterozygous females, the ratio is about 35:65. In addition to the relative activity of the PGK-1 allozymes, we also measured the absolute activity of PGK-1 in oocytes obtained from three types of Xce homozygous females.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental genetics of the gastrulation defective locus in Drosophila melanogaster

The fs(1)gastrulation defective (dg) locus is one of the dorsal-group genes of Drosophila. Maternal expression of this gene is required for gastrulation movements and the differentiation of structures along the embryonic dorso-ventral axis. Twelve alleles of gd displayed a complex pattern of complementation, suggesting a direct interaction between subunits of a multimeric protein. Essential expression of the gd locus was strictly maternal with no zygotic contribution by the paternally derived allele. Clonal analysis revealed that expression of the gd locus was required in the germ line and that extreme dorsalization represented the null gd phenotype. Temperature-sensitive (ts) alleles displayed a ts period that included the last 4-5 hr of oogenesis and the first 1.5-2 hr of embryogenesis. Eggs from one ts allelic combination displayed reduced hatching when retained in the ovary at permissive temperatures, suggesting the loss of a labile egg component. This lability may also be responsible for the variable phenotypes displayed by offspring from individual females.     

Parent-of-origin specific histone acetylation and reactivation of a key imprinted gene locus in Prader-Willi syndrome

To examine the chromatin basis of imprinting in chromosome 15q11-q13, we have investigated the status of histone acetylation of the SNURF-SNRPN locus, which is a key imprinted gene locus in Prader-Willi syndrome (PWS). Chromatin immunoprecipitation (ChIP) studies revealed that the unmethylated CpG island of the active, paternally derived allele of SNURF-SNRPN was associated with acetylated histones, whereas the methylated maternally derived, inactive allele was specifically hypoacetylated. The body of the SNURF-SNRPN gene was associated with acetylated histones on both alleles. Furthermore, treatment of PWS cells with the DNA methyltransferase inhibitor 5-azadeoxycytidine (5-aza-dC) induced demethylation of the SNURF-SNRPN CpG island and restoration of gene expression on the maternal allele. The reactivation was associated with increased H4 acetylation but not with H3 acetylation at the SNURF-SNRPN CpG island. These findings indicate that (1) a significant role for histone deacetylation in gene silencing is associated with imprinting in 15q11-q13 and (2) silenced genes in PWS can be reactivated by drug treatment.     

Perspective: maternal kin groups and the origins of asymmetric genetic systems-genomic imprinting, haplodiploidy, and parthenogenesis

The genetic systems of animals and plants are typically eumendelian. That is, an equal complement of autosomes is inherited from each of two parents, and at each locus, each parent's allele is equally likely to be expressed and equally likely to be transmitted. Genetic systems that violate any of these eumendelian symmetries are termed asymmetric and include parent-specific gene expression (PSGE), haplodiploidy, thelytoky, and related systems. Asymmetric genetic systems typically arise in lineages with close associations between kin (gregarious siblings, brooding, or viviparity). To date, different explanatory frameworks have been proposed to account for each of the different asymmetric genetic systems. Haig's kinship theory of genomic imprinting argues that PSGE arises when kinship asymmetries between interacting kin create conflicts between maternally and paternally derived alleles. Greater maternal than paternal relatedness within groups selects for more "abstemious" expression of maternally derived alleles and more "greedy" expression of paternally derived alleles. Here, I argue that this process may also underlie origins of haplodiploidy and many origins of thelytoky. The tendency for paternal alleles to be more "greedy" in maternal kin groups means that maternal-paternal conflict is not a zero-sum game: the maternal optimum will more closely correspond to the optimum for family groups and demes and for associated entities such as symbionts. Often in these circumstances, partial or complete suppression of paternal gene expression will evolve (haplodiploidy, thelytoky), or other features of the life cycle will evolve to minimize the conflict (monogamy, inbreeding). Maternally transmitted cytoplasmic elements and maternally imprinted nuclear alleles have a shared interest in minimizing agonistic interactions between female siblings and may cooperate to exclude the paternal genome. Eusociality is the most dramatic expression of the conflict-reducing effects of haplodiploidy, but its original and more widespread function may be suppression of intrafamilial cannibalism. In rare circumstances in which paternal gene products gain access to maternal physiology via a placenta, PSGE with greedy paternal gene expression can persist (e.g., in mammals).     

Zn++ stimulation of the induction of alcohol dehydrogenase by anaerobiosis in roots of Zea mays L.

Summary Zn⁺⁺ at an optimum concentration of 5×10^–4 M caused a two fold stimulation in the level of alcohol dehydrogenase (ADH) induced by anaerobic conditions. Isozymes specified by different genes and alleles show disproportionate increases in activity, such that, unequal representation of gene products while not eliminated, is invariably reduced by Zn⁺⁺ treatment. Thus in the case of alleles at the Adh-1 locus, there was a greater increase in the protein subunit specified by the Adh-1S allele. From previous work (Fischer and Schwartz, 1973) this protein is known to have a reduced affinity for Zn⁺⁺. This suggests that zinc availability during ADH induction is limiting and may provide an alternative to the cis-linked regulatory gene model proposed by Schwartz (1971) to explain the unequal expression of genes and alleles.     

A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback

We have developed a non-radioactive in situ hybridization technique for the localization of RNA in whole mount Drosophila embryos. After fixation, whole embryos are hybridized in situ with a DNA probe which has been labeled with digoxygenin. The hybridization products are detected by using a phosphatase-coupled antibody against digoxygenin. In parallel experiments, embryos can be treated with an antibody directed against the corresponding protein product to allow the detection of its distribution using standard immunochemical techniques. We have used this approach to compare the spatial and temporal distribution patterns of the RNA and protein products of the segmentation gene hunchback (hb) during the early stages of embryogenesis. This comparison revealed translational control of the maternally derived hb mRNA, which was difficult to detect by conventional techniques. The non-radioactive in situ hybridization method is as sensitive as conventional methods, but is faster and easier to perform. This may make it a useful tool for a variety of other systems.     

Epigenetic Resetting of a Gene Imprinted in Plant Embryos

Genomic imprinting resulting in the differential expression of maternal and paternal alleles in the fertilization products has evolved independently in placental mammals and flowering plants. In most cases, silenced alleles carry DNA methylation [1]. Whereas these methylation marks of imprinted genes are generally erased and reestablished in each generation in mammals [2], imprinting marks persist in endosperms [3], the sole tissue of reported imprinted gene expression in plants. Here we show that the maternally expressed in embryo 1 (mee1) gene of maize is imprinted in both the embryo and endosperm and that parent-of-origin-specific expression correlates with differential allelic methylation. This epigenetic asymmetry is maintained in the endosperm, whereas the embryonic maternal allele is demethylated on fertilization and remethylated later in embryogenesis. This report of imprinting in the plant embryo confirms that, as in mammals, epigenetic mechanisms operate to regulate allelic gene expression in both embryonic and extraembryonic structures. The embryonic methylation profile demonstrates that plants evolved a mechanism for resetting parent-specific imprinting marks, a necessary prerequisite for parent-of-origin-dependent gene expression in consecutive generations. The striking difference between the regulation of imprinting in the embryo and endosperm suggests that imprinting mechanisms might have evolved independently in both fertilization products of flowering plants.     

Expression of specific genes in early mouse embryos blocked by cytochalasin

Summary Mouse embryos at the two cell stage derived from C57BL/6 × C3H/Aa F₁-females heterozygous at the X-linked phosphoglycerate kinase locus (Pgk-1) were cultured continuously in the presence of cytochalasin B or D. Further cleavage of the two cell embryos was thus prevented and the embryos became polyploid during culture. The onset of expression of the maternally inherited Pgk-1 gene and of the paternally inherited glucosephosphate isomerase (Gpi-1) gene was determined in these polyploid embryos by cellulose acetate gel electrophoresis of single embryos. In contrast to euploid preimplantation embryos developing normally in utero or in culture without cytochalasins, expression of maternal Pgk-1 was never observed at days 4 and 5 of gestation in polyploid two cell embryos, showing that the Pgk-1 allele on the maternally inherited X chromosome is not activated independently of cytokinesis and morphogenesis. Expression of paternally derived Gpi-1, however, occurred in cleavage blocked embryos von day 5 of development. This may indicate that the activation of two genes which are both expressed during preimplantation development and which both code for glycolytic enzymes, is initiated by different signals.     

Wingless mutation inDrosophila melanogaster

A temperature sensitive lethal allele of thewingless locus ofDrosophila melanogaster together with previously studied lethal and viable alleles in this locus, has been used to study some properties of this locus. These studies show the existence of two lethal phases for thewingless lesion; one during embryogenesis and another during pupation. By growing embryos with temperature sensitivewingless lesion at the permissive temperature and letting the larvae develop at non-permissive temperature, a large-scale cell death and subsequent regeneration were seen to occur in the mutant wing discs. This cell death followed by regeneration alters the normal developmental potential of the wing disc. Disc transplantation experiments show that these discs are incapable of differentiating into wing blade structures.